The membrane bound enzyme Na+,K+ ATPase is responsible for the active transport of Na+ and K+ across cell membranes and is, therefore, an essential component of all higher animal cells. It is also the putative receptor site for the cardiac glycosides which are specific inhibitors of its ATPase activity. This enzyme is of special interest because of its role in the maintenance of the electrophysiological properties of heart muscle cells. The goal of this project is to determine the membrane organization, structure, and function of the Na+, K+ ATPase and, thereby, aid in the elucidation of the mechanism of the inotropic effect of the cardiac glycosides. I plan to use antibodies directed against the "native" enzyme and its components in order to determine structure-function relationships. Studies using mouse monoclonal antibodies will investigate specific antibody-enzyme interactions in order to understand the mechanism(s) of antibody effects on enzyme function and the interactions between the different ligand binding sites of the enzyme. In addition, antibodies (polyclonal and monoclonal) with selective specificities towards the holoenzyme, the catalytic, glycoprotein subunit, and specific haptens will be used to determine the influence of the holoenzyme conformation on its antigenic properties and to identify, localize, and isolate functional regions of the enzyme containing the cardiac glycoside receptor, the phosphorylation site, and cation binding sites. This project will also provide the tools and develop the methodologies needed to study the biosynthesis and processing of the catalytic and glycoprotein subunits and to identify the cDNA or chromosomal clones of the catalytic subunit of the Na+, K+ ATPase.